脂多糖对人正常肝细胞株L02损伤的实验研究

目的探讨脂多糖(lipopolysaccharide,LPS)对人正常肝细胞株L02的损伤作用及其机制。方法采用流式细胞术分别检测LPS诱导L02细胞凋亡和线粒体膜电位变化的作用,测定L02细胞膜上CD14、Toll样受体4(TLR4)、Toll样受体2(TLR2)的表达水平;采用酶联免疫吸附法(ELISA)测定细胞培养上清液中肿瘤坏死因子α(TNF-α)含量;生化法测定细胞培养上清液中丙氨酸转氨酶(ALT)、门冬氨酸转氨酶(AST)及乳酸脱氢酶(LDH)含量。结果10、20、40、80mg/L剂量的LPS作用于L02细胞后0、6、12、24和36h,细胞的凋亡率和线粒体膜电位无显著性差异(P>0.05),各组上清液中ALT、AST、LDH和TNF-α含量亦无明显变化(P>0.05),L02细胞膜上LPS受体CD14、TLR4、TLR2表达分别为(2.28±0.60)%,(1.04±0.80)%,(2.07±0.50)%。结论L02细胞膜上LPS受体CD14、TLR4、TLR2表达水平低,致使LPS不能直接引起L02细胞损伤。

An Experimental Study on the Injury of Human Normal Hepatocyte Cell Line L02 by LPS

Objective To observe the effect of LPS on the human normal hepatocyte cell line L02 and investigate the underlying mechanism.Methods The L02 cells were cultured with various concentrations of LPS(at 0,10,20,40 and 80mg/L final concentrations).The cells and culture supernatants were collected after cultured for 0,6,12,24 or 36h.Using flow cytometry technique,we detected the cell apoptosis rate,mitochondria membrane potential alteration and the expressions of CD14,TLR4 and TLR2 on L02 cell membrane.ALT,AST,LDH and TNF-(levels in the supernatants were measured by biochemistry assay and ELISA respectively.Results It was found that CD14,TLR4 and TLR2 molecules were hardly expressed on L02 cell membrane.After stimulation with LPS,the cell apoptosis rate,mitochondria membrane potential,and the enzymes and TNF-(levels had no significant difference(P>0.05)compared with the controls.Conclusions On account of the low expression levels of LPS receptors,CD14,TLR4 and TLR2,L02 cell line is not suitable to for study of LPS on hepatocyte injury.LPS can not cause apoptosis of L02 cell line because of,in part,low expression of LPS receptors on the cell membrane.